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Articles

A bioinspired omniphobic surface coating on medical


devices prevents thrombosis and biofouling
Daniel C Leslie14,9, Anna Waterhouse1,3,5,9, Julia B Berthet1,3,5, Thomas M Valentin1,3, Alexander L Watters1,3,
Abhishek Jain1,3,4, Philseok Kim1,2, Benjamin D Hatton1,2,8, Arthur Nedder6, Kathryn Donovan6,
Elana H Super1, Caitlin Howell1,2, Christopher P Johnson1,2, Thy L Vu1,2, Dana E Bolgen1, Sami Rifai1,
Anne R Hansen3,5, Michael Aizenberg1, Michael Super1,3,4, Joanna Aizenberg1,2,7 & Donald E Ingber14

Thrombosis and biofouling of extracorporeal circuits and indwelling medical devices cause significant morbidity and mortality
worldwide. We apply a bioinspired, omniphobic coating to tubing and catheters and show that it completely repels blood and
2014 Nature America, Inc. All rights reserved.

suppresses biofilm formation. The coating is a covalently tethered, flexible molecular layer of perfluorocarbon, which holds a thin
liquid film of medical-grade perfluorocarbon on the surface. This coating prevents fibrin attachment, reduces platelet adhesion
and activation, suppresses biofilm formation and is stable under blood flow in vitro. Surface-coated medical-grade tubing and
catheters, assembled into arteriovenous shunts and implanted in pigs, remain patent for at least 8 h without anticoagulation.
This surface-coating technology could reduce the use of anticoagulants in patients and help to prevent thrombotic occlusion and
biofouling of medical devices.

Countless lives have been saved by implantable medical devices, because the surface-bound heparin leaches, resulting in a progres-
(artificial hearts, ventricular assist devices, pacemakers, cardioverter- sive loss of anticoagulation activity4,11 and the use of heparin-coated
defibrillators and central lines) and extracorporeal devices that flow materials has not led to a drastic reduction in the clinical use of
whole human blood outside the body through indwelling catheters soluble heparin12. Some high-flow dialysis treatments can be car-
and external circuits during cardiopulmonary bypass, hemodialysis ried out without heparin in subsets of patients with high bleeding
and extracorporeal membrane oxygenation1,2. However, the need risks, but even in this patient population, half are forced to switch
to co-administer soluble anticoagulant drugs, such as heparin, dur- to heparin bolus dialysis within the first year of treatment 2. Due
ing many of these procedures, substantially reduces their safety and to these limitations, other nonthrombogenic, hydrophilic material
hampers their effectiveness3,4. Without systemic anticoagulation, coatings have been explored, including PHISIO (Sorin)13, Trillium
these extracorporeal and indwelling devices can rapidly occlude (Medtronic)14, poly-2-methoxyethyl acrylate (PMEA) polymer15 and
sulfobetaine16. Extensive human clinical evaluation of these vari-
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due to thrombosis because clots form when fibrin and platelets in


the flowing blood adhere to the surfaces of these artificial materi- ous alternative surface coatings is currently underway; however, no
als5. Unfortunately, heparin causes morbidity and mortality through benefit has been shown to date when compared to existing heparin-
post-operative bleeding, thrombocytopenia, hypertriglyceridemia, coated materials17,18.
hyperkalemia and hypersensitivity6, and its use is contraindicated Based on the limited clinical utility of these strategies for reduc-
in several patient populations7. In fact, most drug-related deaths ing thrombosis of extracorporeal circuits, we explored a recently
from adverse clinical events in the United States are due to systemic described, slippery, liquid-infused, porous surface (SLIPS) approach.
anticoagulation8. SLIPS was inspired by the Nepenthes pitcher plant, which uses a layer
The pressing clinical need to prevent blood clotting while mini- of liquid water to create a low friction surface that prevents attach-
mizing administration of anticoagulant drugs has led to the search ment of insects19. The SLIPS technology creates omniphobic slippery
for biomaterial surface coatings that can directly suppress blood clot surfaces by infiltrating porous or roughened substrates with various
formation. The most successful approach to date has been to chemi- liquid perfluorocarbons (LPs) that prevent adhesion to the underly-
cally immobilize heparin on blood-contacting surfaces to reduce ing substrate through formation of a stably immobilized, molecu-
thrombosis and lower anticoagulant administration 9,10. Although larly smooth, liquid overlayer20. However, existing medical-grade
this approach has been widely adopted, major limitations persist materials, such as polycarbonate, polysulfone and polyvinyl chloride

1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA. 2School of Engineering and Applied Sciences, Harvard
University, Cambridge, Massachusetts, USA. 3Harvard Medical School, Boston, Massachusetts, USA. 4Vascular Biology Program, Boston Childrens Hospital, Boston,
Massachusetts, USA. 5Division of Newborn Medicine, Boston Childrens Hospital, Boston, Massachusetts, USA. 6Animal Research, Boston Childrens Hospital,
Boston, Massachusetts, USA. 7Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. 8Present address: Department of
Materials Science and Engineering, University of Toronto, Ontario, Canada. 9These authors contributed equally to this work. Correspondence should be addressed to
D.E.I. (don.ingber@wyss.harvard.edu).

Received 6 November 2013; accepted 13 August 2014; published online 12 October 2014; doi:10.1038/nbt.3020

1134 VOLUME 32 NUMBER 11 NOVEMBER 2014 nature biotechnology


Articles

(PVC), have highly smooth surfaces. Thus, to create nonadhesive, relevant plastics, glasses and metals, including polycarbonate, PVC,
antithrombogenic surfaces that might be useful for clinical medicine polysulfone, polyethylene, polypropylene, polyethylene terephtha-
in the near-term, we set out to modify the SLIPS technology so that late (PET), polyimide, polystyrene, borosilicate glass, polydimethyl-
it can be applied to these smooth surfaces. This was accomplished siloxane (PDMS), titanium, silicon, fluorinated ethylene propylene
by covalently binding a flexible molecular perfluorocarbon layer, and polytetrafluoroethylene (Fig. 1d and Supplementary Fig. 2). The
or tethered perfluorocarbon (TP), on the material surface and then TLP-coated smooth surfaces containing a thin, molecular film of LP
coating it with a mobile layer of an LP (perfluorodecalin) that has immobilized through interactions with a covalently coupled TP layer
been used extensively in medicine for applications such as liquid were also equally as effective as roughened or porous materials into
ventilation21,22, ophthalmic surgery23 and as an US Food and Drug which a larger volume of LP was passively infused, as described in
Administration (FDA)-approved blood substitute24 (http://www.fda. the original SLIPS coating technology; SLIPS-coated expanded PTFE
gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInform (e.SLIPS) and nanostructured boehmite (B.SLIPS) (Fig. 1d) have pre-
ation/Guidances/Blood/ucm074920.htm). We refer to this antithrom- viously been shown to repel crude oil and ice, as well as anticoagulated
bogenic bilayer composed of the TP with an LP coating as a tethered- animal blood20,25,26. Additionally, the TLP-coated, smooth, medical-
liquid perfluorocarbon (TLP) surface. Here, we show that the TP grade materials showed no blood adhesion after the greater challenge
retains the free LP as a thin mobile liquid layer applied to virtually any of immersion in human blood, which was in stark contrast to the
medical-grade material surface, even when the surface is in contact control (Supplementary Movie 3).
with a flowing, immiscible fluid, such as blood (Fig. 1a). Importantly, To further illustrate how slippery the TLP coating is, we tested the
the TLP surface coating also effectively repels whole blood, resists ability of TLP-coated acrylic to repel the adhesion of a living gecko,
adhesion of blood components and bacteria, and reduces thrombosis which was previously shown to be unaffected by surface coatings on
in vitro and in vivo without dangerous anticoagulants. acrylic27. The stable adhesion of the LP to the TP is maintained by van
der Waals attractive forces. Thus, we tested whether the gecko, which
2014 Nature America, Inc. All rights reserved.

RESULTS evolved to provide maximum van der Waals forces over 17 times their
TLP surface coating repels whole blood body weight on a vertical surface27, could overcome the TLP coating.
To test the antiadhesive properties of the TLP coating method, weex- Geckos remained attached to control acrylic surfaces oriented at up
amined surface adhesion of fresh whole human blood on an acrylic to 90 degrees inclination (Supplementary Movie 4); however, the
surface with or without a TLP coating (tethered perfluorohexane and geckos were unable to hold on when the acrylic surface was coated
liquid perfluorodecalin) that was sloped at an angle of 30 degrees. with TLP, and thus, they slipped down before the angle approached
Blood droplets immediately adhered to the control uncoated acrylic vertical (Supplementary Movie 5).
surface and left a trail of blood components over the time course of 5 s
(Fig. 1b, top, Supplementary Fig. 1 and Supplementary Movie 1). In Reduced adhesion and activation of blood components
contrast, when the same surface was coated with TLP, the blood drop- Material-induced thrombosis is mediated through adhesion and
let almost immediately slid off the surface (<0.3 s), and, remarkably, activation of two major blood components, fibrinogen and platelets,
there was no evidence of any residual blood trail (Fig. 1b, bottom,
Supplementary Fig. 1 and Supplementary Movie 2). We quantified a
blood adhesion to surfaces by measuring the minimum angle required LP

to cause a droplet to slide (sliding angle) (Fig. 1c). Control uncoated Blood

surfaces and surfaces that were modified by coating with either the
TP or LP layer alone all exhibited considerable blood adhesion, even
when the surface was tilted to 90 degrees (Fig. 1c). In contrast, the
npg

minimum angle to cause blood sliding on the TLP acrylic surface TP


Substrate
was 0.6 0.2 degrees. Importantly, this same TLP surface coating was Sliding angle
equally effective when applied to multiple other smooth medically
b 0s 2s 5s

Figure 1 TLP-coated surfaces repel whole human blood. (a) Schematic Control
of blood repellency on TLP surfaces showing the TP bound to a substrate
through plasma activation and silane treatment, which then allows a
stable film of LP to adhere to the surface. (b) Surfaces without TP or
LP (TP/LP; control) show adhesion of a blood droplet (50 l, 3.2% 0s 0.2 s 0.3 s
sodium citrate) on the 30-degree angled surface, low velocity and
residence over 5 s (upper panels). When TLP is applied to the surface,
a blood droplet (50 l, 3.2% sodium citrate) is immediately repelled and TLP
slides down the surface at an incline of 30 degrees within 0.3 s (lower
panels). Scale bars, 1 cm. (c) Graph showing the minimum angle that
allowed whole blood (5 l droplet, 3.2% sodium citrate) to slide on the
different surface treatments (mean s.d., n = 3). (d) TLP can be applied c 90
d 90
to a wide range of materials with a low whole-blood sliding angle (black
Sliding angle

Sliding angle

bars) compared to control surfaces (gray bars) comprising polycarbonate


(degrees)

(degrees)

60 60
(PC), PVC, polysulfone (PSU), polyethylene (PE), polypropylene (PP),
polyethylene terephthalate (PET), polyimide (PI), polystyrene (PS), 30 30

borosilicate glass (G), titanium (Ti), silicon wafer (Si), fluorinated ethylene 0
0
propylene copolymer (FEP), polytetrafluoroethylene (PTFE), expanded LP: + +
PVC
PSC
U
PE

PEP
T
PI
PS
G
Ti
F i
eP EP

B. LIPE
SL S
S
S
P

e. TF

IP
P

polytetrafluoroethylene (ePTFE), Slippery Liquid Infused Porous Surface TP: + +


S

(SLIPS) (e.SLIPS) and boehmite SLIPS (B.SLIPS) (mean s.d., n = 3). Surface treatment Surface treatment

nature biotechnology VOLUME 32 NUMBER 11 NOVEMBER 2014 1135


Articles

Figure 2 Whole blood interactions with TLP surfaces. (a) Fluorescent a Control TP TLP
micrographs of acrylic (upper panels) or polysulfone (lower panels)
pieces (11 8 mm) after 90-min incubation with fresh human blood

Acrylic
containing heparin (0.25 U/ml) and fluorescent fibrinogen (150 g/ml)
showing polymerized fibrin networks on the control (left), decreased
network formation on TP (middle) and punctate staining with minimal
network formation on TLP (right). Scale bars, 50 m. (b) Graph showing a

Polysulfone
reduction of percent fibrin-covered area on TLP (full line) acrylic compared
to control (dotted line) and TP (dashed line). Percentages quantified
using ImageJ (*P < 0.05 compared to control, two-way ANOVA, s.e.m.).
(c) Graph showing a reduction of percent fibrin-covered area on TLP (full
line) polysulfone compared to control (dotted line) and TP (dashed line) b Acrylic c Polysulfone

Fibrin clot (% area)

Fibrin clot (% area)


60 60
(*P < 0.05 compared to control, two-way ANOVA, s.e.m.). (d,e) Graph
showing a 27-fold reduction in platelet adhesion (number per 150 m2) on 40 40
TLP acrylic compared to control (d) and about fourfold reduction on TLP *
* *
polysulfone (e) (*P < 0.05, one-way ANOVA, s.e.m.). (f) Scanning electron 20 20 * *
micrographs of acrylic surfaces after 30-min incubation shows reduced * *
0 0 *
platelet adhesion on TLP compared to control and TP. Scale bars, 10 m.
30 60 90 30 60 90
Insets show platelet morphology, white arrowheads show platelets. Inset Time (min) Time (min)
scale bar, 2 m. All data are from experiments with three separate donors,
two technical replicates, eight (b,c) or four (d,e) images per replicate. d 200 Acrylic e 150 Polysulfone

Adherent platelets
Adherent platelets
150
within minutes of surface contact. Cleavage of fibrinogen to fibrin 100

results in the formation of polymerized fibrin networks that form 100


2014 Nature America, Inc. All rights reserved.

the backbone of the clot, which becomes intertwined with aggregates 50


50
of platelets that are simultaneously activated by either interacting *
directly with the material surface or with surface-bound proteins5. 0 * 0
Control TP TLP Control TP TLP
We exposed smooth plastic surfaces to fresh, whole human blood that
contained a low dose of heparin (0.25 U/ml) to prevent immediate f Control TP TLP

clotting (compared to 57 U/ml clinical dosage). Even in the presence


of heparin, control acrylic and polysulfone surfaces formed dense
fibrillar networks of polymerized fibrin during the 90-min study
(Fig. 2a). Whereas surfaces coated with a TP layer alone (without
the LP layer) reduced network formation, adherent fibrils of polymer-
ized fibrin were still detected. In contrast, coating the acrylic surface
with the TLP bilayer completely prevented adhesion of fluorescently Suppression of thrombosis under flow in vitro
labeled fibrin in whole blood. Similarly, TLP-coated polysulfone To be medically relevant, the TLP surfaces must remain stable and
showed only minimal punctate fibrin staining with rare thin fibrils functional when exposed to physiological shear stresses. When we
(Fig. 2a). Quantitation of these results confirmed that TLP coating of exposed the surfaces to a constant shear strain rate (1,000 s 1) using a
the acrylic and polysulfone substrates decreased fibrin adhesion and rheometer, we found that LP was displaced from acrylic surfaces that
polymerization relative to controls by six- and fourfold, respectively were coated with LP alone after 1 min of exposure to shear, resulting
(P < 0.05), and by three- and twofold relative to surfaces coated with in blood adhesion even when the surface was tilted to 90 degrees. In
npg

the TP layer alone (Fig. 2b,c). The reduced adhesion and polymeriza- contrast, TLP-coated acrylic surfaces continued to effectively repel
tion of fibrin on the surfaces coated only with the TP layer is impor- blood (sliding angle < 3 degrees) even after 16 h exposure to the same
tant because it suggests that TLP coatings will significantly suppress amount of fluid shear (Fig. 3a). Also, these TLP surfaces were stable
clot formation, even if the LP layer is displaced after extended expo- when exposed to a range of shear rates (2502,000 s1) for 10 min with
sure to blood. Furthermore, we confirmed the reduced coagulation no change in sliding angle (Supplementary Fig. 5). Thus, the TLP
on TLP surfaces exposed to whole blood by showing that D-dimer coating remained stable under hemodynamic fluid shear conditions
production, which is a quantitative marker of fibrin polymerization, for extended time periods in vitro because of the higher affinity of LP
was significantly (P < 0.05) lower on the TLP coating than on control for the immobilized TP than for blood or water.
surfaces (Supplementary Fig. 3). We then flowed fresh whole human blood (0.25 U/ml heparin at
To evaluate effects on platelet adhesion, we analyzed surfaces by 50 ml/h) through either control or TLP-coated medical-grade PVC
scanning electron microscopy (SEM) after they were exposed to whole tubing (1/16 inner diameter (ID)). After rinsing, the remaining adher-
human blood for 30 min. These studies revealed that coating acrylic ent proteins were digested off the tubing surface and quantified using
and polysulfone surfaces with TLP reduced platelet adhesion by 27- a bicinchoninic acid (BCA) assay (Fig. 3b). These studies revealed
and fourfold, respectively, compared to control surfaces (P < 0.05, that coating the tubing with TLP resulted in a threefold decrease
Fig.2d,e). Furthermore, although nearly all platelets adherent to con- in protein adhesion compared to control tubing (0.8 0.2 versus
trol materials and surfaces coated with TP alone exhibited a flattened 2.3 0.5 g/cm2, respectively; P < 0.05) (Fig. 3b). Free hemoglobin
or spread morphology consistent with platelet activation, most platelets levels were not significantly different from those measured in baseline
found on surfaces coated with TLP were round, suggesting that they blood samples, indicating that the TLP surfaces caused no hemolysis
remained in a resting, nonactivated state (Fig. 2f and Supplementary with a single pass of flowing blood (Supplementary Fig. 6).
Fig. 4). Taken together, these results indicate that TLP surfaces reduce We then challenged the TLP coating by testing it in a microfluidic
fibrin polymerization and suppress both adhesion and activation of device to mimic the physiological microenvironment of blood flowing
platelets, hence confirming their reduced thrombogenicity in vitro. through a vessel. Fresh whole human blood (0.5 U/ml heparin) was

1136 VOLUME 32 NUMBER 11 NOVEMBER 2014 nature biotechnology


Articles

a 90 b 4 c 1.0 d 40 e 100 * f g
* *
Sliding angle (degrees)

Thrombus weight (mg)


80

Platelets remaining
Relative flow rate
3 30

Protein adhesion

in blood (%)
60
60

(g/cm2)
2 0.5 20 Control
40
30
1 10
20

0 0 0 0 0
LP 0.1 1 10 100 1,000 0 10 20 30 40 50

l
P

l
P
TLP Control TLP

tro

tro

tro
TL

TL

TL
Time (min) Time (min)

on

on

on
C

C
Figure 3 Stability and thrombogenicity of TLP surfaces. (a) The low blood sliding angle on TLP acrylic discs (black bars) remained stable
over 1,000 min under a constant shear rate of 1,000 s 1, whereas LP acrylic discs (gray bar) failed and had a 90-degree sliding angle after 1 min
(s.d.). (b) Decreased protein adhesion on TLP PVC, measured by BCA assay, after whole human blood flow (0.25 U/ml heparin) through 1/16 ID PVC
tubing for 20 min at 50 ml/h. (*P < 0.05, paired students t-test, s.d.) (c) Maintenance of whole human blood flow (0.5 U/ml heparin at 1,250 s 1) in
TLP microfluidic channels (PDMS) over 50 min (black line), whereas LP channels occluded after ~15 min (gray line). (d) Reduced thrombus weight
in TLP medical-grade PVC tubing (black bar) compared to control (gray bar, *P < 0.05, unpaired, two-tailed students t-test, s.d.) after blood was
pumped in a closed loop at 3 L/h for 2 h. (e) Increased percentage of platelets remained in the blood after exposure to TLP tubing (black bar)
compared to control tubing (gray bar, *P < 0.05, unpaired, two-tailed students t-test, s.d.) after the blood was pumped in a closed loop at 3 L/h for 2 h.
(f) Photographs of filtered thrombi after blood was pumped in a closed loop at 3 L/h for 2 h in control (upper image) and TLP medical-grade PVC
(lower image). Scale bars, 5 mm. (g) Photographs of control (left) or TLP (right) cardioperfusion tubing sterilized with ethylene oxide after it had been
exposed to porcine blood for 2 min. Scale bars, 5 mm. All data are from experiments with three separate donors.
2014 Nature America, Inc. All rights reserved.

infused under a constant pressure with an initial wall shear of 1,250 s1. perfusion tubing (Supplementary Fig. 9b), and ethylene oxide steri-
Blood flowed without occlusion for longer in TLP devices than in lization of the TP-coated perfusion tubing did not alter the surfaces
LP devices (Fig. 3c), as shown by a significant 2.5-fold increase ability to repel blood when coated with LP before use (Fig. 3g). This
in average clotting time (P < 0.05), which corresponds to the time coating method therefore provides an effective method to create anti-
required for the occlusion to reduce flow by half (Supplementary thrombogenic surfaces on existing medical-grade materials, which is
Fig. 7). This indicates that overall thrombosis was significantly amenable to rapid commercial translation.
reduced on TLP surfaces, as we expected from the reduced fibrin
polymerization and suppressed adhesion and activation of platelets Reduced thrombosis in vivo
that we observed in our earlier studies (Fig. 2). To examine the antithrombogenic properties of TLP-modified, medical-
To explore whether this surface-coating technology might be useful grade materials in vivo, commercially available polyurethane
for extracorporeal circuits in the clinic, the TLP coating was applied to cannulae, polycarbonate connectors and PVC cardiopulmonary
large-diameter (1/4 ID), medical-grade PVC cardioperfusion tubing. perfusion tubing were assembled into an arteriovenous (AV) shunt
No macroscopic or microscopic differences were observed with the and modified with the TLP surface coating. We then analyzed its
application of TP to this tubing (Supplementary Fig. 8a,b). Distinct ability to support blood flow for 8 h in a porcine femoral AV shunt
droplets of blood could also be seen moving along the TLP surface model (Fig.4a), either with low heparin (30 U/kg compared to the
without adhesion, whereas blood adhered to surfaces that were coated 300 U/kg used for conventional systemic heparinization28) or without
with LP alone (Supplementary Fig. 8c). As extracorporeal circuits any systemic heparin anticoagulation. In the low heparin group, real-
require peristaltic pumps to drive blood flow, we pumped fresh whole time flow measurements revealed that flow rates remained between
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human blood (0.25 U/ml heparin) at 3 liters (L)/h through a closed 10 and 21 L/h in animals implanted with either uncoated control or
loop (1/4 ID perfusion tubing) system linked with polycarbonate TLP-modified shunts, although we observed increased flow rate vari-
connectors. These studies showed that there was greatly reduced ability in animals with the control (noncoated) materials (Fig.4b).
formation of visually detectable clots within the lumen of the tub- Importantly, all of the TLP-coated circuits also remained patent for
ing in the TLP-coated PVC loops (Fig. 3d,f). This correlated with 8 h even in the absence of any heparin, maintaining a flow rate of 11
a reduction in thrombus weight by about fourfold after 2 h com- to 18 L/h; in contrast, complete occlusion occurred in four out of five
pared to uncoated control loops (P < 0.05, Fig. 3d), and the finding uncoated circuits in control animals (P < 0.05; Fig. 4c). In animals
that blood platelet counts remained about threefold higher in blood that did not receive heparin, two animals in the TLP group and one
exposed to TLP-coated loops than uncoated loops (P < 0.05, Fig. 3e) is in the control group developed similarly low mean arterial pressures.
indicative of reduced platelet adhesion and/or aggregation. Although Nevertheless, despite the increased thrombogenic potential from these
there was some loss (28 21%) of platelets in this experiment, the decreased flow rates, the circuits remained patent in the TLP animals,
blood traveled through the pump 360 times, the equivalent of pass- whereas the circuit became fully occluded in the control animal.
ing through 150 m of tubing. This is far harsher than extracorpor- Analysis of circuits from both heparin and no-heparin groups
eal circulation, and hence, a similar result is not to be expected to after completion of the study revealed that TLP-modified circuits
occur in vivo. Importantly, although the use of peristaltic pumps can exhibited a marked decrease in occlusive thrombosis in both the arte-
sometimes mechanically abrade the tubing where they interface, this rial and venous sections after 8 h of flow (Fig. 4d). Total thrombus
did not impair the antithrombogenic properties of the TLP surface. weight in TLP-coated circuits was reduced by twofold compared to
Furthermore, the TLP coating was consistent along the entire length controls in the heparin and no-heparin groups (P < 0.05; Fig. 4e).
of a 120 (10 ft.) segment of cardioperfusion tubing (Supplementary To determine the extent of occlusion throughout the circuits post-
Fig. 9a), as verified by similar tilt angles and a high percentage of explant, we measured lumen areas using computerized image analysis
fluorine atoms throughout (Supplementary Table 1). We also found to calculate percent occlusion. In the heparin group, control circuits
that the TP was stable for 12 months when applied to cannulae and had 7 3% occlusion, whereas there was no detectable occlusion

nature biotechnology VOLUME 32 NUMBER 11 NOVEMBER 2014 1137


Articles

Figure 4 Thrombogenicity of TLP-coated circuits in a porcine a Arteriovenous


e
arteriovenous shunt model. (a) Schematic of the porcine arteriovenous shunt model
15 *
shunt model showing placement of the cannulae in the femoral artery

Thrombus weight (g)


and vein connected by tubing. Black arrows indicate direction of blood
10
flow driven by the heart. (b) Real-time measurements of flow rate Aorta
(10 s average) at the midpoint of the circuit showing that in heparinized Femoral artery
animals (+ heparin), flow rate is maintained for 8 h in both control 5
Cannula
(gray lines, n = 3) and TLP (black lines, n = 2). (c) Kaplan-Meier curve of
patency from real-time flow-rate measurements in nonheparinized Tubing
animals ( heparin) revealed 4 out of 5 control circuits (gray line, n = 5) Femoral vein 0
+ Heparin Heparin
occlude, whereas TLP circuits (black line, n = 4) remain patent for 8 h
(*P < 0.05, Fishers exact test). (d) Photographs of polyurethane
cannulae, polycarbonate connectors and PVC tubing of TLP (top) and b 30
+ Heparin
f 50
control (bottom) circuits after 8 h of blood flow. Arrows indicate direction *
of blood flow through arterial (Art) or venous (Ven) cannula. Increased 24 40

Flow rate (L/h)

Occlusion (%)
thrombus is visible in the control circuit. Scale bars, 5 mm. (e) Thrombus 18 30
weight in the TLP circuits (black bars) is reduced under both + heparin
and heparin conditions compared to control circuits (gray bars) 12 20
(*P < 0.05, two-way ANOVA with Bonferronis multiple comparisons test,
6 10
s.e.m.). (f) Percent occlusion of circuits showing that TLP (black bar)
has minimal occlusion compared to control (gray bars) in the presence or 0 0
absence of heparin (*P < 0.05, two-way ANOVA with Bonferronis multiple 0 2 4 6 8 + Heparin Heparin
comparisons test, s.e.m.). (g) Biofilm formation on control PVC (gray Time (h)
bars) and TLP PVC (black bars) for 1.5 (*P < 0.05, two-way ANOVA with c Heparin g
2014 Nature America, Inc. All rights reserved.

100 15 *
Bonferronis multiple comparisons test, s.e.m.) and 6.5 weeks. (h) Crystal

P. aeruginosa (CFU 103)


TLP
violetstained P. aeruginosa on polyethylene control (left) and TLP (right) 80
surfaces after culturing for 16 h. Scale bars, 5 mm. Insets show SEM 10
Patency (%)

images. Scale bars, 2 m. 60

40
5
Control
in circuits coated with TLP (Fig. 4f and Supplementary Fig. 10). 20 *
Moreover, even in the absence of heparin, there was a 2.5-fold reduc- 0 0
tion in lumen occlusion (from 35 8% to 14 4%) in TLP-coated 0 2 4 6 8 1.5 6.5
Time (h) Time (weeks)
circuits compared to controls (P < 0.05; Fig. 4f and Supplementary
Fig. 10). These data confirm our in vitro results by showing that the d Art Cannula Connector Tubing h Control TLP
TLP coating significantly reduced the thrombogenicity of medical- Ven TLP
grade plastics, including widely used PU, polycarbonate and PVC,
Art
when in direct contact with whole blood flowing at high, physiologi-
Ven Control
cally relevant rates (1021 L/h) for at least 8 h in vivo. Importantly, in
these studies, the TLP coating was as effective at maintaining shunt
blood flow with no heparin as control circuits in heparinized animals surface with LP using the SLIPS technology30. Thus, the TLP coat-
(Fig. 4b,c). Blood flow was also maintained without producing sig- ing not only displayed antithrombogenicity, it also exhibited potent
nificant differences in hematologic parameters, pathology or histol- antibiofouling capabilities. Other antibiofouling surface modification
npg

ogy (Supplementary Note and Supplementary Table 2), and there methods (e.g., sulfobetaine modification) have also been shown to
was minimal leaching of LP into the blood. In the few blood samples reduce microbial attachment and biofilm formation in vitro, but only
where it was detected (4 of 19 samples from three animals), free LP over the course of 24 h16.
levels were at or near the limit of detection (0.07 g/ml blood or
~4 g LP/kg body weight), which is over six orders of magnitude DISCUSSION
lower than levels of LP that have been used clinically without toxicity The TLP coating that we described in this study can be applied to a
(5.6 g LP/ kg body weight)24,29. wide range of different, commercially available, medical-grade mate-
rials to create surfaces that are both antithrombogenic and antibio-
TLP coating inhibits biofilm formation fouling. TLP-coated surfaces resisted adhesion of fibrin and platelets,
Given the potent ability of the TLP coating to prevent surface adhe- suppressed biofouling and reduced thrombosis when the surfaces
sion, we also explored whether this surface-coating method could came in contact with flowing whole human blood driven by a peri-
prevent adhesion of microorganisms and subsequent biofilm forma- staltic pump in vitro. Importantly, TLP-coated AV shunts also retained
tion on medical materials, which can lead to clinical infections and their ability to prevent occlusive thrombus formation for at least 8 h
increased risk of sepsis in patients with indwelling medical devices. in vivo when implanted as a flowing vascular circuit in pigs.
In fact, when Pseudomonas aeruginosa bacteria were grown in TLP- Sulfobetaine surface coatings have been previously shown to reduce
coated loops of PVC medical tubing for up to 6.5 weeks, there was an thrombus formation when applied to medical-grade materials in the
eightfold reduction in biofilm formation compared to control tub- presence of slightly reduced heparin concentrations (0.7 to 1 U/ml
ing (P < 0.05) (Fig. 4g). The TLP coating also suppressed biofouling compared to 57 U/ml used clinically). When a sulfobetaine-coated
on other materials (PET and acrylic), as indicated by significantly catheter was inserted into a canine jugular vein in the absence of
reduced bacterial adhesion of P. aeruginosa and Escherichia coli rela- anticoagulants for 4 h, clot formation was reduced on its outer surface,
tive to control surfaces (P < 0.05; Fig. 4h and Supplementary Fig. 11); but the surface of the inner lumen was not studied16. Sulfobetaine
these results are similar to those obtained by infusing a roughened surface coatings also have not been shown to withstand mechanical

1138 VOLUME 32 NUMBER 11 NOVEMBER 2014 nature biotechnology


Articles

pumping. In contrast, in our in vivo model, arterial blood flowed for medical devices and implants with complex shapes because the
through the lumen of the surgically implanted shunt tubing under plasma permeates tortuous paths and surface features down to the
a much higher pressure and shear rate (ten times higher than in the microscale40. This is a great advantage relative to other surface coat-
canine venous model), and thrombosis was reduced for twice as long ings, such as sulfobetaine, which uses a peroxide to activate the surface
(8 h versus 4). In addition, in our in vitro studies, the TLP coating that can generate bubbles, resulting in some areas being left untreated
prevented coagulation in blood containing one-fourth the heparin that can become thrombogenic41.
dose used in the sulfobetaine coating study (0.25 U/ml versus 1 U/ml), This TLP coating represents the first surface coating to reduce
and we did this in blood flow circuits driven by a peristaltic pump, thrombosis under physiological arterial flow in vivo without the use
which is crucial for clinical medical devices that use extracorporeal of soluble anticoagulants, and in tubing that experiences peristaltic
circuits (gravity flow was used in the other studies). In addition, we pumping ex vivo using reduced levels of anticoagulation (0.25 U/ml
successfully created antithrombogenic surfaces by applying the TLP heparin). Thus, it could be employed to coat materials used for vari-
coating to 20 different, medically relevant biomaterials, whereas the ous types of extracorporeal circuits, and it might potentially be valu-
sulfobetaine coating method for thrombus reduction has only been able for coating indwelling devices, including total artificial hearts
demonstrated with polyurethane16. Importantly, the TLP process is and ventricular assist devices, as well as needles, Vacutainers, sutures
the first nonheparin coating to show reduced thrombogenicity in vivo and blood storage bags. Importantly, although there are commercially
on the inner lumen of tubing, which is most clinically relevant for use available surface-coating technologies that partially reduce either
in extracorporeal circuits. blood thrombosis or bacterial adhesion, the TLP coating prevents
The reduction in thrombosis we obtained with the TLP-coated both. Because the TLP coating technology also prevents biofouling,
circuit is similar to that obtained with other heparin-based coat- it opens up the possibility of creating a new class of medical materials
ings in porcine and bovine cardiopulmonary bypass models, but and devices lined by antithrombogenic and antibiofouling surfaces
heparin-based coatings can leach into blood and require exposure of that do not require co-administration of antiplatelet, anticoagulant or
2014 Nature America, Inc. All rights reserved.

the blood to extremely high heparin doses (>50 U/kg)31,32. The TLP antibiotic medications when implanted in patients. This would reduce
coating more closely resembles silicone liquid thin films, which were the need for systemic heparinization and antibiotic drug treatments to
previously used in vitro to delineate blood coagulation mechanisms prevent related morbidity and fatalities, which would greatly decrease
without anticoagulants for short durations33. However, silicone liquid healthcare costs.
thin films do not prevent platelet adhesion or activation34 and have
not been shown to reduce thrombosis in vivo. Hydrophobic surfaces Methods
(e.g., ePTFE, which is a porous perfluorinated solid material) are Methods and any associated references are available in the online
clinically approved as vascular grafts35; however, these fail to improve version of the paper.
the performance of medical devices36 because they contain trapped
Note: Any Supplementary Information and Source Data files are available in the
air that can be thrombogenic5.
online version of the paper.
To our knowledge, there is no other known medical material
coating that can effectively suppress occlusive thrombosis in vivo Acknowledgments
under high pressure and high-shear arterial flow in the complete This work was supported by Defense Advanced Research Projects Agency grant
N66001-11-1-4180 and contract HR0011-13-C-0025, and the Wyss Institute for
absence of heparin. Thus, as the TLP coating does not leach anti-
Biologically Inspired Engineering at Harvard University. We thank D. Super,
coagulant activity into the blood, it could potentially offer a new R. Cooper, E. Murray and J. Lee for phlebotomy, T. Ferrante for assistance with
way to prevent thrombosis without the complications of heparin fluorescence microscopy, H. Kozakewich for assistance with histology evaluation
anticoagulant therapy. and O. Ahanotu for assistance in preparing surfaces. Scanning electron microscopy
The TP we used was covalently coupled to the surface by the same and X-ray photoelectron spectroscopy were conducted at the Center for Nanoscale
npg

Systems at Harvard University, a member of the National Nanotechnology


silane chemistry that is used in dental adhesives, but other reactive Infrastructure Network, which is supported by the National Science Foundation
groups could be used to generate TP layers25. The LP perfluorodecalin (ECS-0335765).
used here has been previously included in an FDA-approved blood
substitute; however, we have found that a variety of LPs, medical grade AUTHOR CONTRIBUTIONS
D.C.L., A.W., J.B.B., T.M.V., A.L.W., A.J., M.A., M.S., J.A. and D.E.I. designed the
and nonmedical grade, can also repel blood when integrated into
research. D.C.L., A.W., J.B.B., T.M.V., A.L.W., A.J., P.K., B.D.H., E.H.S., D.E.B. and
a TLP coating (Supplementary Fig. 12). An important caveat for S.R. performed experiments. D.C.L., A.W., J.B.B., T.M.V., A.L.W., A.J., E.H.S., M.A.,
future clinical application of this technology is to avoid use of LPs M.S., J.A. and D.E.I. analyzed data. C.H., C.P.J., T.L.V., M.A. and J.A. designed,
that evaporate at body temperature, which can induce gaseous emboli performed and analyzed the PFD leaching study. D.C.L., A.W., J.B.B., A.N., K.D.,
formation and considerable toxicity37. Additionally, it is likely that D.E.B., A.R.H., M.S. and D.E.I. designed, performed and analyzed the in vivo
study. D.C.L., A.W., A.L.W., M.A., M.S., J.A. and D.E.I. wrote the paper with
the surfaces with the TP layer will be sterilized and stored dry, and input from all authors.
then the LP will be added to the circuit shortly before use, allowing
blood to flow through these TLP-coated circuits. This is consistent COMPETING FINANCIAL INTERESTS
with the saline priming step that is currently used clinically with The authors declare no competing financial interests.
extracorporeal circuits, and so it should be easily integrated into Reprints and permissions information is available online at http://www.nature.com/
these protocols. reprints/index.html.
Another advantage of the TLP coating method is that it relies on
the use of a low-pressure plasma surface modification procedure
1. McCarthy, P.M. & Smith, W.A. Mechanical circulatory supporta long and winding
commonly used for commercial modification of materials38. This road. Science 295, 998999 (2002).
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15891602 (2013).
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as the plasma occurs at approximately ambient temperature38, and open heart operations. Ann. Thorac. Surg. 58, 391398 (1994).

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1140 VOLUME 32 NUMBER 11 NOVEMBER 2014 nature biotechnology


ONLINE METHODS 10 scans. XPS confirmed the highly fluorinated surface layer (TP) on acrylic,
Modification of surfaces with TP. Samples were briefly exposed (40 s, polycarbonate, polysulfone and PVC tubing (Supplementary Table 1).
unless stated otherwise) to low-pressure (150 to 250 mTorr) radio-frequency
(13.56 MHz) oxygen plasma at 100 Watts in order to gently activate the sur- Gecko adhesion. Study approval was obtained from Boston Childrens Hospital
face to react with the TP silane in a PE-100 plasma system (PlasmaEtch). Institutional Animal Care and Use Committee (protocol number 13-10-2552).
Immediately following plasma activation, samples were immersed in a liquid One eyelash-crested (Rhacodactylus ciliatus) gecko was placed inside an acrylic
silane solution (5% v/v tridecafluoro-1,1,2,2-tetrahydrooctyl trichlorosilane cylinder and tilted slowly from horizontal to vertical. This was repeated with
(Gelest, Morrisville, PA) in anhydrous ethanol (Sigma, St. Louis, MO)) for the gecko inside a TLP-treated acrylic cylinder and the tilting was stopped
1 h at room temperature. Treated samples were rinsed with anhydrous ethanol when the gecko slid to the bottom of the cylinder. This was repeated a total of
(Sigma, St. Louis, MO), distilled, deionized water (Milli-Q Water Purification three times with the gecko allowed to recuperate for 1 week between experi-
System, Millipore), and three times with pure ethanol (Koptec, King of ments. Videos are representative of the three trials with the same results.
Prussia, PA). Rinsed samples were gently blown dry with compressed nitro-
gen and gently heated in an oven with desiccant at 60 C overnight at atmos- Surfaces under shear. Surfaces were exposed to shear using a rheometer
pheric pressure. Medical-grade cardiopulmonary perfusion tubing (Sorin (TA Instruments, Model AR-G2) with a 40-mm diameter, 2-degree angle
Group, Arvada, CO) was exposed to oxygen plasma for 3 min; 8 French (Fr) cone-and-plate setup (TA Instruments, Model 513406.905) and Peltier plate
pediatric arterial cannulae (polyurethane and polycarbonate connectors) (TA Instruments, Model 531051.901). Acrylic sheets were cut into 40-mm
(Bio-Medicus, Medtronic, Minneapolis, MN), monitoring lines (Smiths diameter discs using a laser cutter (Epilog Legend 36EXT). These discs were
Medical, St. Paul, MN) and four-way stopcocks (Smiths Medical, St. Paul, MN) then aligned with the cone platen, and stuck to the bottom Peltier plate platen
were exposed to oxygen plasma for 2 min. Other materials treated with oxygen with adhesive (3M, St. Paul, MN). The samples were then lubricated with just
plasma for 40 s were: poly (methyl methacrylate) (PMMA), polysulfone (PSU), enough PFD to cover the surface of the acrylic disc (~500600 l of PFD).
polypropylene (PP), polytetrafluoroethylene (PTFE), polyethylene terephtha- Approximately 2 ml of 35% v/v glycerol (Sigma, St. Louis, MO) in water was
late (PET), ultra-high molecular weight polyethylene (UHMW PE), polycar- applied on top of the lubricated acrylic disc. The cone platen was then low-
bonate (Goodfellow, Coraopolis, PA), polystyrene (BD Biosciences, Durham, ered 50 m above the acrylic disc, and the excess PFD and glycerol was then
2014 Nature America, Inc. All rights reserved.

NC), PVC, fluorinated ethylene propylene (FEP), perfluoroalkoxy (PFA), pushed out from between the platens. This excess fluid was removed to allow
titanium, polyimide, stainless steel (McMaster-Carr, Robbinsville, NJ), glass a meniscus of glycerol solution to form between the two platens. Solvent rings
cover slip (VWR, Radnor, PA), polydimethylsiloxane (Dow Corning, Midland, were then placed around the setup to minimize evaporation of the liquids.
MI), Aeos HyFlex expanded polytetrafluoroethylene (ePTFE) (Zeus), silicon For studies with shear rates below 500 s1, a 10-min conditioning step was
wafer (Ted Pella, Redding, CA). Atomic force microscopy revealed minimal performed at 500 s1 to ensure the excess PFD was removed.
change in surface roughness after TP coating (3.4 1 nm) compared to control
acrylic (2.0 0.2 nm) (mean s.d., n = 3). Scanning electron microscopy. Samples that had been in contact with blood
or bacteria were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate
Sliding angle of surfaces. The angle at which a droplet of liquid begins to move buffer (Electron Microscopy Sciences, Hatfield, PA) for 1 h, 1% osmium
across a surface (sliding angle) was measured for the samples using a manual tetroxide in 0.1 M sodium cacodylate (Electron Microscopy Sciences, Hatfield,
goniometer (Thor Labs GN05/M). Samples were dip-coated in perfluorodeca- PA) for 1 h, dehydrated in ascending grades of ethanol, and chemically dried
lin (PFD) (FluoroMed, APF-140HP (sterile, high purity), Round Rock, TX) with hexamethydisilazane (Electron Microscopy Sciences, Hatfield, PA) in a
immediately before measurement and the sample was placed on top of the lev- desiccator overnight before being mounted and sputter-coated with a thin layer
eled goniometer. The amount of PFD on the surface was 46 l/cm2 as meas- of gold/palladium and imaged on a Zeiss Supra55VP microscope.
ured by an analytical balance after dip coating. For tilting at 30 degrees, 200
l of citrated whole human blood (see Human blood samples11) was placed Human blood samples. Approval for studies involving human subjects was
gently onto surfaces in ~50 l droplets. For quantification of sliding angle, obtained from Harvard University Faculty of Medicine Committee on Human
a 5-l droplet of citrated whole human blood was gently placed on the surface. Studies (protocol number M20403-101). Whole human blood was obtained
The sample was then tilted until the droplet was observed sliding along the sur- from healthy volunteers with informed consent who were nonsmokers and
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face. For samples that did not slide by 15 degrees, a custom built setup smoothly had not taken aspirin for the 2 weeks before donation. Blood was drawn by
tilted the sample to 90 degrees. Samples still adherent at 90 degrees were noted standard venipuncture into no additive Vacutainers (Becton Dickenson).
as a sliding angle of 90 degrees. Sliding angle measurements were obtained on A discard tube was drawn first, then heparin (1,000 U/ml) (APP
TLP acrylic with alternate LPs: perfluorohexane (PFH, Sigma), perfluorooctane Pharmaceuticals, Schaumburg, IL) was added to subsequent tubes to a final
(PFO, Sigma), 1-bromoperfluorooctane (PFOB, Oakwood Products), perfluoro concentration dependent on the assay. Assays were based on ISO-10993-4 for
perhydrophenanthrene (Vitreon, FluoroMed APF-215HP (sterile, high purity)), evaluation of medical devices42.
perfluorotripentylamine (FC-70, HamptonResearch), perfluorotributylamine/
perfluorodibutylmethylamine (FC-40, Santa Cruz Biotechnology), 3-ethoxy- Whole blood adhesion assay. Wells of a 24-well plate were blocked with 1% (w/v)
1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-trifluoromethyl-hexane (HFE-7500, 3M), bovine serum albumin (BSA) (Sigma A3803, St. Louis, MO) in saline for
perfluoropolyether oils (Krytox 101, Krytox 103, food grade Krytox FG-40, 30 min and rinsed with saline. Samples (11 8 mm) of 100-m thick poly
Dupont). sulfone or 1/16-thick acrylic were control, TP or dip coated in PFD to gen-
erate TLP. Samples were incubated in blocked wells with heparinized whole
Elemental analysis methods. X-ray photoelectron spectroscopy (XPS) was human blood (0.25 U/ml to prevent immediate clotting while retaining the
performed on a Thermo Scientific K-Alpha X-Ray Photoelectron Spectrometer ability of blood components to be activated by surfaces). Sample order was
(Thermo Scientific). Samples were prepared with TP treatment as described randomized for incubation with blood. Fluorescently labeled fibrinogen
above. PVC tubing was stored under vacuum before XPS analysis to accelerate (150 g/ml, Invitrogen, Carlsbad, CA, 90% clottable fraction) was added to
outgassing of plasticizers. Auto-Analysis scans (XPS displacement tolerance the blood, which was then incubated with samples for increasing time points
of 8eV, Auger displacement tolerance of 12eV) were performed and analyzed on an orbital shaker. D-dimer concentration was measured in blood from one
using the Thermo Scientific Avantage Data System v5.915 (Thermo Scientific). donor by enzyme-linked immunosorbent assay (ELISA) after 60 min (Sekisui
Three TP samples and one control sample were tested. Three points were Diagnostics, LLC, Stamford, CT) according to the manufacturers instructions.
selected on each sample with a spot size of 30 m. Five survey scans (binding Acrylic and polysulfone samples were washed three times in normal saline
energy range from 4 to 1350 eV) were averaged at each point to identify (0.9% sodium chloride; Baxter Healthcare, Cambridge, MA) and fixed for
potential elements on the sample surface. Based on the potential elements 1 h with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer before imag-
identified during the survey scans, higher resolution individual elemental ing with a Hamamatsu 9100-02 EMCCD camera on a Zeiss Axio Observer
analysis scans were then performed for each elemental range and averaged over Z1 fluorescent microscope using a 20 objective and Metamorph software.

doi:10.1038/nbt.3020 nature biotechnology


Quantification was carried out using ImageJ (http://imagej.nih.gov/ij/) on collected into EDTA Vacutainers for CBC analysis at the time of venipuncture,
eight unadjusted images from each sample. The images from each experiment and again after being pumped through the tubing and filtered through the cell
were converted to binary images and generated into a stack. The threshold was strainer. Reported values of the percentage of platelets remaining in blood were
manually set to 6 for all images, and the area covered by fibrin was extracted calculated as the platelets in the sample collected after pumping divided by
from the measurements. A maximum fibrin clot area of 6070% was quanti- the platelets in the sample collected before tubing multiplied by 100%. Each
fied due to the fibrillar nature of the thrombus on the surface and imaging experiment was done with three donors.
on the surface focal plane. Each experiment was done with two replicates per
donor and repeated with three donors. Representative images in Figure 2a Porcine arteriovenous shunt model. Study approval was obtained from
were converted to green and brightness and contrast was uniformly adjusted Boston Childrens Hospital Institutional Animal Care and Use Committee
to all for clarity. Samples from the 30-min time point were processed for SEM (protocol number 12-06-2202) and conducted in an AAALAC-accredited
as described above. Scanning electron micrographs from four areas per sam- USDA registered facility. A total of 15 female Yorkshire swine weighing
ple were obtained, and a blinded researcher counted the number of adhered 2435 kg (34 months old) were used in this study. Animals were randomly
platelets. Nonactivated platelets were differentiated from activated platelets by assigned to control or TLP groups (no blinding was performed as this was
their round or dendritic morphology, as defined by Goodman43. Nonactivated not possible due to the application of LP immediately before implantation of
platelets were subsequently counted, identified by rounded morphology with the shunt). No power analysis was performed to determine sample size. One
zero flattened protrusions. The number of nonactivated platelets was divided group of animals received 30 U/kg heparin at the time of circuit placement
by total adherent platelets per field of view, and multiplied by 100% to obtain (n = 3 control and n = 2 TLP). One animal in the TLP group was excluded
the percent activated platelet value. as the TLP treatment was performed outside the prespecified parameters
specified above; the pressure gauge on the plasma system was found to be
Blood flow in small-diameter medical-grade tubing. TP monitoring lines out of calibration. The second group received no heparin (n = 5 control and
(1/16 ID by 48 length) were primed with 3 ml of PFD. Control and TLP tub- n = 4 TLP). The control group received unmodified extracorporeal materials
ing were subsequently primed with saline. Human heparinized whole blood and the TLP group received an extracorporeal circuit in which all the materials
(0.25 U/ml) at room temperature was drawn through the monitoring line at a had been treated with TP, sterilized with ethylene oxide and lubricated with
2014 Nature America, Inc. All rights reserved.

flow rate of 60 ml/h by a syringe pump (Harvard Apparatus, Holliston, MA) 10 ml sterile LP (as received from Fluoromed) and drained within 10 min
by withdrawal. This flow rate corresponds to a shear strain rate of 40 s1 and before implantation. The amount of PFD on the circuits at implantation was
was maintained over 20 min. Blood was collected into a 20-ml syringe (Becton 0.81.2 g.
Dickinson, Franklin Lakes, NJ) containing a 1:9 volume of 3.8% sodium cit- Animals were anesthetized with intramuscular injections of atropine
rate (Ricca Chemical Company, Arlington, TX). Tubing was rinsed with 20 (0.04 mg/kg), telazol (4.4 mg/kg) and xyalzine (2.2 mg/kg) and maintained
ml normal saline at a flow rate of 60 ml/h. Blood samples were collected into on isoflurane (1.52.5%) and oxygen (1.2 liter/min) delivered through a
EDTA vacutainers (Becton Dickinson, Franklin Lakes, NJ) for 18-parameter 7-mm endotracheal tube using a positive pressure ventilator. The animal was
complete blood count using the VetScan HM2 Hematology System (Abaxis, placed in the supine position and a 6 Fr percutaneous sheath catheter was
Union City, CA). Tubing was incubated with buffer composed of 0.04 U/ml placed in the left femoral artery for pressure monitoring. A 20-g intravenous
plasmin (Hematologic Technologies Inc., Essex Junction, VT), normal saline, cannula was placed in the left marginal ear vein for administration of drugs
6.25 mM CaCl2, and 4.68 mM MgCl2 for 1 h at 37 C. Samples were then cen- and a 10 Fr Foley catheter was placed for urinary drainage. The AV shunt was
trifuged at 200 g for 10 min to sediment red blood cells, and the total protein established via cutdown of the right femoral artery and vein and insertion of
concentration was measured in the supernatant by a BCA protein assay fol- 8 Fr pediatric arterial cannulae (Bio-Medicus, Medtronic, Minneapolis, MN).
lowing manufacturers protocol (Thermo Fisher Scientific, Waltham, MA). The cannulae were connected to the extracorporeal circuit, which consisted
The mass of protein was then divided by the area of the inner lumen of the of 25 of ID perfusion tubing (Sorin Group) with a 1/4 barbed connector
tubing (60.8 cm2) to determine the protein adsorbed/cm2. Further, we mea (Sorin Group) and large bore four-way stopcock (with rotating male luer lock,
sured adsorption of physiological levels of bovine serum albumin (50 mg/ml) Baxter) placed 3 from the venous cannula. The perfusion tubing was filled
to test the hypothesis that this assay reflects the adhesion of abundant plasma with 1 U/ml heparinized saline during placement, which was fully drained
proteins that are less relevant to thrombus formation. We detected very small before establishing circuit flow. Shunt implantation time was greater than two
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amounts of albumin bound to the surface (0.035 0.002 g/cm2) that were times the activated clotting time. The heart rate, pulse rate, arterial pressure,
barely above background levels. Each paired (TLP versus control) experiment oxygen saturation, CO2 level, temperature and respiratory rate were monitored
was done with three donors. throughout the experiment. Vitals were maintained at physiological levels and
body temperature was maintained at 37 C by means of a heat mat. After 8 h,
Blood flow in microfluidic channels. TP-treated and control microfluidic animals were given 300 U/kg heparin and euthanized with an intravascular
PDMS microdevices with microchannels 200 m wide by 75 m tall were lethal dose of Fatal Plus.
primed with LP. Human heparinized whole blood (0.5 U/ml) was flowed in
the constant pressure mode with a calculated initial wall shear rate of 1,250 s1 In vivo blood sampling and flow measurements. At baseline (before AV
(50 dynes/cm2) by means of a syringe pump (PHD Ultra CP, Harvard cannulation), time 0 h (immediately after arteriovenous circuit flow was
Apparatus) with disposable pressure sensor (PendoTECH). Clotting times established), time 3.5 h, and time 7.5 h, blood samples (20 ml) were taken for
were derived by finding the time for flow to reduce to half its initial value, from complete blood count (CBC), blood gas and chemistry profiles, and clotting
a sigmoidal decay fitted curve (n = 3 donors). Because the microfluidic device times (PTT, APTT, ACT) (EDTA, heparin and citrate Vacutainers, respectively).
required more extensive time for setup, we used a slightly higher heparin dose Flow was measured in the midpoint of the perfusion tubing using a clamp-on
to prevent coagulation before TLP exposure before the analysis was carried tubing flow sensor connected to a TS410 flow meter module (Transonic, Ithaca,
out. This dose (0.25 U/ml) was still very low in comparison to that commonly NY) for 15 min after circuit flow was established and every 30 min thereafter for
used in standard in vitro coagulation assays (5 to 15 U/ml). ~15 min each time. Occlusion was measured post-explant by taking photographs
of cross-sections of the cannulae and tubing and digitally determining the area
Blood flow in large diameter medical-grade tubing. TLP PVC tubing (1/4 of the lumen and area of the thrombus to calculate the percent occlusion.
ID by 15 length) was primed with 10 ml of PFD. Control and TLP tubing
were subsequently washed with saline. PVC tubing was connected with con- Histology. Organ samples (lung, liver, kidney, spleen and brain) were fixed in
trol or TLP 1/4 ID polycarbonate barbed connectors and filled with human 10% neutral buffered formalin (Electron Microscopy Sciences, Hatfield, PA)
heparinized whole blood (0.25 U/ml). Blood was pumped at a flow rate of for 24 h at room temperature. Tissue was processed and stained with
3 L/h (shear rate of 250 s1) by a peristaltic pump (Cole Parmer, MasterFlex hematoxylin and eosin (H&E) at Boston Childrens Hospital Histopathology
L/S, Vernon Hills, IL) for 2 h. Blood was filtered through a pre-weighed 40 m services. No evidence of thrombi or microemboli was found in either control
cell strainer and air-dried before thrombus weight was obtained. Blood was or TLP lung sections.

nature biotechnology doi:10.1038/nbt.3020


Gas chromatography/mass spectrometry (GC/MS). Blood samples detection of P. aeruginosa on tubing segments. Tubing was washed in Tris-
(2 ml) were extracted with methyl nonafluoroisobutylether (HFE) (Miller- buffered saline, 0.1% Tween 20 (TBS-T) (Boston BioProducts, Ashland,
Stephenson) and analyzed by gas chromatography/mass spectrometry44. MA) supplemented with 5 mM CaCl2 (Boston BioProducts, Ashland, MA),
The limit of detection was determined by multiplying the s.d. of the baseline followed by incubation with FcMBL-HRP in 3% BSA in TBS-T 5 mM CaCl2.
response by 3; this was converted into a clinical dose by multiplying the limit The colorimetric reaction was done with the Pierce 1-Step TMB substrate
of detection (LOD) by 60 ml of blood/kg of body weight. (Thermo Fisher Scientific, Waltham, MA) according to the manufacturers
protocol, and absorbance at 450 nm was measured. Bacterial titers were estab-
Biofouling assay. Control and TP samples of acrylic (11 8 mm, 1/16 thick) lished by comparison to a standard curve of P. aeruginosa. The culture removed
were sterilized with ethanol (pure ethanol, 200 proof) and allowed to air dry from the loops after 6.5 weeks was confirmed to be viable by plating.
in a biological laminar flow hood for 30 min. Samples were transferred to
24-well plate wells and sterile LP was added to TP samples. LP was removed Statistical analysis. Data are expressed as mean s.d. for Figures 1 and 3 and
and all samples were immediately incubated with 105 CFU of E. coli (ATCC mean s.e.m. for all other data. In vitro assay sample size was predetermined
8739) in RPMI (Invitrogen, Carlsbad, CA) for 48 h at 37 C. After culture, with three separate donors to account for biological variability. The in vivo
samples were washed three times in PBS and assayed for biofilm formation patency data are shown as a Kaplan-Meier curve (Fig. 4c). Data were sta-
using crystal violet (Becton Dickenson, Sparks, MD). Samples were incubated tistically analyzed by paired, two-tailed students t-test (Fig. 3b), unpaired,
with 0.1% crystal violet for 1 h and washed six times with distilled water. The two-tailed students t-test (Fig. 3d,e and Supplementary Figs. 3,7,11), Fishers
crystal violet was solubilized with 10% acetic acid for 10 min before 100 l exact test (Fig. 4c), one-way analysis of variance (ANOVA) (Fig. 2d,e and
was transferred to a 96-well plate and the absorbance measured using a plate Supplementary Fig. 4) and two-way analysis of variance (ANOVA) with
reader at 590 nm. The inoculum and cultures of the control and TLP samples Bonferroni post-hoc analysis for multiple comparison (Fig. 2b,c and 4eg).
after 48 h were confirmed to be viable by plating. P < 0.05 was considered significant (as indicated in the figures by an asterisk),
PE and PET samples (1 1) were incubated with P. aeruginosa (a clinical although caution is necessary due to the small sample sizes and therefore the
isolate from Brigham and Womens Specimen Bank (protocol number M20403- normality assumption cannot be tested. Paired analysis was only used for
101)) overnight. Samples were washed in PBS, stained with crystal violet for experiments that used blood from a single venipuncture on both control and
2014 Nature America, Inc. All rights reserved.

15 min, washed with distilled water three times and photographed or fixed for experimental surfaces. Prism version 6.00 (GraphPad Software) was used for
SEM. Bacterial adhesion under continuous flow was tested using a modified statistical analysis.
Chandler loop setup. The P. aeruginosa bacteria were cultured in RPMI Media
(Life Technologies, Carlsbad, CA) at 37 C. Control and TLP loops (3-mm
inner diameter) were filled with P. aeruginosa cultures (105 CFU/ml) and
incubated at 25 C. 1.5 weeks and 6.5 weeks after initial inoculation, two 1-cm 42. Biological Evaluation of Medical Devices, Part 4: Selection of Tests for Interactions
with Blood, 2002, Second Edition and 2006 Amendment ISO 10993-4 (Geneva,
segments of tubing were assayed for biofilm formation. To measure bacterial
International Standards Organization, 2006).
adhesion on the tubing surface we used a novel FcMBL fusion protein45. Briefly, 43. Goodman, S.L. Sheep, pig, and human plateletmaterial interactions with model
the carbohydrate recognition domain of Mannose Binding Lectin (MBL) was cardiovascular biomaterials. J. Biomed. Mater. Res. 45, 240250 (1999).
fused to the Fc domain of human IgG and recombinantly expressed in Chinese 44. Audran, M. et al. Determination of perfluorodecalin and perfluoro-N-
methylcyclohexylpiperidine in rat blood by gas chromatographymass spectrometry.
hamster ovary cell lines. FcMBL was conjugated with horseradish peroxidase
J. Chromatogr. B Biomed. Sci. Appl. 745, 333343 (2000).
(FcMBL-HRP) using the Lightning Link-HRP Antibody Labeling Kit (Novus 45. Kang, J.H. et al. An extracorporeal blood cleansing device for sepsis therapy.
Biologicals, Littleton, CO) and used as a detection antibody for ELISA-based Nat. Med. doi:10.1038/nm.3640 (14 September 2014).
npg

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